KR20160042254A - Process for preparing lipase derived from Candida antarctica using Pichia pastoris - Google Patents

Abstract

The present invention relates to a mutated lipase produced by transforming lipase derived from a Candida antarctica strain, a polynucleotide encoding the mutated lipase, an expression vector including the polynucleotide, a transformant into which the expression vector is introduced, a method for producing the mutated lipase by using the transformant, and a method for producing biodiesel using the mutated lipase. The use of the mutated lipase gene of the present invention enables the production of the lipase in excellent efficiency from the transformant derived from the Pichia pastoris strain. Thus, the method can be widely used in the biological production process for various compounds using the lipase, including the biodiesel production method.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a process for preparing a lipase derived from Candida antacitica strain using a Pichia pastoris strain,

The present invention relates to a method for producing a lipase derived from Candida antigorosa strain using a Pichia pastoris strain, and more particularly, to a method for producing lipase derived from Candida antica, A method for producing the mutated lipase using the transformant, and a method for producing the biodiesel using the mutated lipase, which comprises the steps of: And a method of producing the same.

The market for bio-catalytic enzymes is showing rapid growth despite showing a ripple effect in various industries. There are about 130 kinds of industrial enzymes and new functional enzymes, but in the actual enzyme market, less than 30 enzymes account for more than 90%. Therefore, in order to utilize the advantages of high efficiency, high precision and high selectivity of biocatalyst, it is required to develop clean bioconversion technology that can replace pollution inducing and high energy requirement chemical synthesis reaction.

One of these enzymes for biocatalysis, lipase (glycerol ester hydrolases), refers to enzymes that catalyze the hydrolysis or synthesis of a wide range of water-soluble or water-insoluble carboxylic acid esters or amides Catalyzed reactions such as hydrolysis, alcoholysis, acidolysis, esterification, and aminolysis, and are active in aqueous as well as organic solvents. And exhibits various characteristics such that it does not require a separate cofactor in the catalytic reaction process, exhibits substrate specificity for various substrates, and has isomer selectivity that is active only for one of the optical isomers, Industrial utility has been proven. In addition to hydrolysis of an ester bond, it also has a function of synthesizing an ester, which is a reverse reaction of the ester bond. Thus, a variety of biocatalysts for producing various chemical synthetic materials comprising ester bond with raw materials and additives such as foods, medicines and cosmetics using triglycerides Utilization is being developed.

For example, the lipase not only plays an important role in lipid metabolism in vivo, but also acts to enhance cheese flavor, increase free fatty acid in vegetable fermentation, improve aroma in meat ripening and decompose fish fat, and produce pure optical isomer synthesis And can be used in a wide range of applications are known to have. In the field of dairy industry, it is used for the production of cheese through decomposition of milk fat and the fat decomposition of butter and cream. In the field of detergent industry, it is used for washing or washing detergent, , Reduction of reaction temperature, etc. In the oil chemical industry, it is used for production of unsaturated fatty acid, soap, and cocoa butter using cheap palm oil. In the field of paper industry, In the field of cosmetics industry, it is used in the production of wax, skin products, tanning cream, bath products, etc. In the field of energy industry In the field, it is being used for the production of biodiesel from vegetable oil.

Such lipases are known to be produced from various animals, plants and microorganisms. Recently, a method of mass-producing lipase using a bioreactor using genetic engineering techniques has been developed. In particular, a method of expressing lipase on the cell surface of single cell organisms such as bacteria and yeast including bacteriophage, a method of simultaneously producing various industrially useful biocatalysts by expressing various kinds of lipases from a single microorganism, And secretory production methods are being developed. For example, Korean Patent Registration No. 475133 discloses a method for screening mutant lipases using a yeast surface expression vector, and Korean Patent No. 658193 discloses a novel lipase and lipase exhibiting a low-temperature lipolytic activity and it is disclosed a method of mass production this, Korea Patent Publication No. 2009-0065673 discloses a Yalova subtotal repository for urticae (Yallowia lipolytica yeast strain is cultured, and an alkaline lipase is obtained from the culture solution thereof.

Among the above-described methods, a method of producing secreted lipase by using yeast has received attention. E. coli, which has been used as a main host cell of a conventional genetic engineering bioreactor, is capable of relatively easily expressing genes derived from various animals, plants and microorganisms, has a high rate of cell growth, and has a low probability of gene mutation in the host However, since the expression efficiency of the introduced gene is low, it is difficult to carry out the post-transcriptional processing step and it is not easy to secrete the expressed protein from the introduced gene, so that it can not be applied to industrial production . On the contrary, the yeast has an excellent expression efficiency of the introduced gene as compared with Escherichia coli, can be processed after the decoding, and can easily produce the secreted protein from the introduced gene, Since the technology level is accumulated at a similar level and industrial production using the introduced gene is easy, a method of secretion production of lipase using yeast is attracting attention. However, it is not easy to express the introduced foreign gene as compared with Escherichia coli. Therefore, the nucleotide sequence of the gene to be introduced should be optimized according to the yeast strain to be used. However, since the industrial productivity is excellent, Studies are underway to develop nucleotide sequences of transgenes that can be optimized.

Under these circumstances, the present inventors have found that a yeast strain, Pichia pastoris ), which has been found to be optimal for the expression of lipase gene, Candida antakica ( Candida antarctic acid strains were used, the lipase expression was optimized, and the present invention was completed.

One object of the present invention is to provide a mutated lipase wherein the lipase derived from the Candida antichatica strain is mutated.

It is another object of the present invention to provide a polynucleotide encoding said mutated lipase.

It is still another object of the present invention to provide an expression vector comprising the polynucleotide.

It is still another object of the present invention to provide a transformant into which the above expression vector has been introduced.

It is still another object of the present invention to provide a method for producing the mutated lipase using the transformant.

It is still another object of the present invention to provide a method for producing biodiesel using the mutated lipase.

The present inventors have found that a yeast strain, Pichia pastoris) to develop a lipase gene can be expressed in these strains optimized results of various studies, Candida aentak Utica (Candida antarctica strains were mutated so as to optimize the expression in P. falciparum strains. The mutated lipase is composed of an amino acid sequence which differs in two parts compared to a lipase derived from a Candida antatica strain. The polynucleotide encoding the mutated lipase is characterized in that the wild-type lipase is expressed in a Pichia pastoris strain It was confirmed that it could be effectively expressed in the Pichia pastoris strain.

Therefore, it was expected that the use of the polynucleotide encoding the mutated lipase provided by the present invention could be used for the efficient production of lipase using the Pichia pastoris strain.

In one embodiment for achieving the above object, the present invention provides a mutated lipase comprising the amino acid sequence of SEQ ID NO: 3.

The term "lipase " of the present invention refers to an enzyme that catalyzes the hydrolysis or synthesis of a wide range of water-soluble or water-insoluble carboxylic acid esters or amides, including hydrolysis, alcoholysis, acidolysis, esterification, aminolysis, and the like.

The lipase maintains its activity not only in an aqueous solution but also in an organic solvent and does not require a cofactor for the catalytic reaction and exhibits substrate specificity for various substrates and is active only for one of the optical isomers Which is one of the industrially applicable enzymes. To date, many kinds of animals, plants and microorganisms have been reported to produce lipase. Studies on the biochemical characteristics of lipase and studies for obtaining lipase gene are underway. Particularly, researches are being actively carried out to utilize lipase throughout the industry such as foods, detergents, fine chemicals, cosmetics and alternative energy industries. In particular, it is known that lipase plays an important role in the field of fine chemicals synthesizing high-value-added leading substances including medicines, because chiral materials widely used in industry contain a lot of ester bonds. Most medicines and pesticides with chirality are important for producing only active enantiomers, since only certain enantiomers have pharmacological activity and other enantiomers have no activity or even toxicity. Up to 700 psi Extreme conditions are not required when lipase is used as compared with the asymmetric chemical synthesis method requiring extreme conditions such as high pressure and high temperature of 250 DEG C or more, so that productivity can be improved.

In the present invention, Candida (SEQ ID NO: 3) which is obtained by mutating two amino acids in the wild type lipase having the amino acid sequence of SEQ ID NO: 1 derived from an antarctic strain. When the amino acid sequence (SEQ ID NO: 1) of the wild type lipase and the amino acid sequence (SEQ ID NO: 3) of the mutated lipase were compared, two amino acids (A57T and T89A) were different from the total 317 amino acids and 99.4% , The mutated lipase containing the amino acid sequence of SEQ ID NO: 3 is not yet known, and thus the mutated lipase provided by the present invention can be said to be the first to be identified by the present inventors.

In addition, as long as the mutated lipase provided by the present invention can be more efficiently expressed in the Pichia pastoris strain than the conventional lipase, various amino acid sequences may be added to the N-terminal or C-terminal of the amino acid sequence of SEQ ID NO: And may include all of the added peptides. In addition, it may further comprise a targeting sequence, a tag, an amino acid sequence designed for specific purposes to increase the stability of the labeled residue, half-life or peptide.

In addition, if the amino acid sequence of the amino acid sequence of SEQ ID NO: 3 is mutated by a method such as addition, substitution, deletion, or the like, as long as it can be more efficiently expressed in the Pichia pastoris strain than the conventional lipase, May be included in the category of mutated lipase.

In particular, the mutated lipases provided herein may comprise a polypeptide having a sequence that differs from the amino acid sequence of SEQ ID NO: 3 by one or more amino acid residues. Amino acid exchange in proteins and polypeptides that do not globally alter the activity of the molecule is known in the art. The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In addition, it may include a protein having increased structural stability or increased protein activity due to mutation or modification of the amino acid sequence, for example, heat, pH, etc. of the protein.

Finally, the mutated lipase provided by the present invention can be prepared by a chemical peptide synthesis method known in the art, or the polynucleotide encoding the mutated lipase can be amplified by PCR (polymerase chain reaction) or synthesized by a known method And then cloning into an expression vector for expression.

As another embodiment for achieving the above object, the present invention provides a polynucleotide comprising a base sequence encoding the mutated lipase.

In the present invention, the nucleotide sequence constituting the polynucleotide is preferably such that the nucleotide sequence (SEQ ID NO: 2) of the polynucleotide capable of encoding the amino acid sequence (SEQ ID NO: 1) of the wild-type lipase is effectively expressed in the Pichia pastoris strain The nucleotide sequence encoding the optimized nucleotide sequence (SEQ ID NO: 4) or the various amino acid sequences that may be added to the N-terminal or C-terminal of the amino acid sequence of SEQ ID NO: 3 is capable of encoding the amino acid sequence of SEQ ID NO: (SEQ ID NO: 4) of the polynucleotide having the nucleotide sequence shown in SEQ ID NO: 3, preferably a nucleotide sequence of SEQ ID NO: 4 capable of coding the amino acid sequence of SEQ ID NO: 3 Or a nucleotide sequence encoding various amino acid sequences that may be added to the N-terminal or C-terminal of the amino acid sequence of SEQ ID NO: 3 May be added to the 5'-terminal or 3'-terminal of the nucleotide sequence of SEQ ID NO: 4.

The nucleotide sequence of SEQ ID NO: 4 was greatly changed when compared with the nucleotide sequence of SEQ ID NO: 3 encoding wild-type lipase, but the amino acid sequence was changed to only two out of 317 nucleotides, Without changing the amino acid, it was expected that the nucleotide could be optimized to be more efficiently expressed in the Pichia pastoris strain.

Since the nucleotide sequence of the polynucleotide encoding the mutated lipase provided in the present invention is not known yet in comparison with the nucleotide sequence of the polynucleotide encoding lipase which has been known so far, the polynucleotide of the same base sequence in nature has not yet been known, The polynucleotide provided by the present invention can be said to be the first to be identified by the present inventors.

In addition, as long as it is capable of encoding a mutated lipase that can be more efficiently expressed in the Pichia pastoris strain than the conventional lipase, a polynucleotide including a base sequence having homology with the above base sequence is also provided The polynucleotide may be a polynucleotide including a nucleotide sequence having a homology of 80% or more, more preferably a polynucleotide comprising a nucleotide sequence having 90% or more homology , And most preferably a polynucleotide comprising a nucleotide sequence that exhibits at least 95% homology.

On the other hand, the polynucleotide may be mutated by substitution, deletion, insertion, or a combination of one or more bases. When the nucleotide sequence is prepared by chemically synthesizing, there can be used a method well known in the art, for example, a method described in Engels and Uhlmann, Angew Chem IntEd Engl., 37: 73-127, 1988 , Triesters, phosphites, phosphoramidites and H-phosphate methods, PCR and other auto primer methods, and oligonucleotide synthesis on solid supports.

In another embodiment for achieving the above object, the present invention provides an expression vector comprising the polynucleotide.

The term "expression vector" of the present invention means a gene construct comprising a gene insert that is capable of expressing a desired protein in an appropriate host cell, and an essential regulatory element operably linked to the expression of said gene insert. The expression vector comprises expression control elements such as an initiation codon, a termination codon, a promoter, an operator, etc. The initiation codon and termination codon are generally considered to be part of the base sequence encoding the polypeptide, and when the gene product is administered, And must be in coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

The term "operably linked" of the present invention means a state in which a nucleic acid sequence encoding a desired protein or RNA is functionally linked to a nucleic acid expression control sequence so as to perform a general function . For example, a nucleic acid sequence encoding a promoter and a protein or RNA may be operably linked to affect the expression of the coding sequence. The operative linkage with an expression vector can be produced using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.

In the present invention, the expression vector may be prepared by introducing a polynucleotide encoding the mutated lipase provided by the present invention into a strain of Pichia pastoris to be a transformant capable of producing a lipase in which the Pichia pastoris strain is mutated Can play a role of mutating.

To this end, the expression vector may comprise a signal sequence for the release of the mutated lipase to facilitate the separation of the protein from the cell culture. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include the ATG start codon, should be provided. These exogenous translational control signals and initiation codons can be of various natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcription or translation enhancers.

In addition, the expression vector may further comprise a protein tag that can be removed using an endopeptidase, optionally in order to facilitate detection of the mutated lipase provided herein.

The term "tag " of the present invention means a molecule exhibiting quantifiable activity or property and includes a polypeptide fluorescent substance such as a fluorophore such as fluorescein, a fluorescent protein (GFP) Or may be a fluorescent molecule including; Myc tag, a Flag tag, a histidine tag, a Lucin tag, an IgG tag, and a strap tagidine tag. Particularly, in the case of using an epitope tag, a peptide tag composed of preferably 6 or more amino acid residues, more preferably 8 to 50 amino acid residues can be used.

As another embodiment for accomplishing the above object, the present invention provides a transformant wherein the expression vector is introduced into a host cell.

The transformant provided in the present invention is produced by introducing the expression vector provided in the present invention into a host cell and transforming the polynucleotide contained in the expression vector to produce the mutated lipase of the present invention Can be used.

The host cell into which the expression vector provided in the present invention can be introduced is not particularly limited as long as it is capable of producing the peptide by expressing the polynucleotide. However, the host cell may be any of E. coli, Streptomyces, Salmonella typhimurium Bacterial cells such as; Yeast cells such as Saccharomyces cerevisiae, ski survey caromyces pombe, and picia paspolus; Insect cells such as Drosophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, Bowmanella cells; Or plant cells, and preferably can be a Pichia pastoris strain.

The term " Pichia " pastoris strain "means yeast strains which are most widely used for the production of recombinant proteins together with Saccharomyces cerevisiae . They are easy to genetically manipulate, have various expression systems developed, and are easy to mass culture As well as post-translational modification functions of proteins such as secretion function and sugar chain addition that can secrete the protein out of the cell when recombinant production of high protein derived proteins such as human protein Secretion production of recombinant protein is possible by extracellular secretion by artificially fusing the protein secretion signal and the target protein. Through protein secretion process, protein folding, disulfide bond formation and sugar chain addition The process is progressive and thus has a biologically complete activity Recombinant proteins can be produced. Moreover, since proteins having biological activity can be obtained directly from the medium, it is very economical since economically inefficient cells are not required to be pulverized or refolded.

Further, the transformant may be performed by various methods, though not one capable of producing a lipase mutant according to the present invention showing the effect which can improve a variety of cellular activity at a high level, specifically limited to, CaCl ₂ precipitation method, a CaCl ₂ precipitation to Hanahan method with improved efficiency by using a reducing substance of DMSO (dimethyl sulfoxide), electroporation (electroporation), the calcium phosphate precipitation method, protoplast fusion method, a stirring method using a silicon carbide fiber, Agrobacterium bacteria Mediated transformation, transformation using PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods.

Meanwhile, the transformant provided in the present invention is prepared by introducing the expression vector into a host cell, Pichia pastoris strain. In the produced transformant, the production efficiency of the mutated lipase provided by the present invention is increased A step of screening the transformant may be further performed, and the resulting transformant may be obtained. Unlike the bacteria in which the expression vector exists as an independent gene and each cell expresses the same introduced gene, even if the same expression vector is introduced into the yeast strain Pichia pastoris, the gene introduced into each transformant May be different. Thus, screening for selecting the transformants most efficiently producing CalB-OPT among the transformants can be carried out to obtain transformants having increased production efficiency of the mutated lipases provided by the present invention have.

Thus, the inventors in the yeast strain Pichia pastoris strains, Candida aentak urticae (Candida antarctica ), and a gene coding for a novel lipase containing the amino acid sequence of SEQ ID NO: 3 was introduced into a transformant. This transformant was named " Pichia pastoris KCM-AT ( Pichia pastoris KCM-AT) "and deposited it with the accession number KCTC18326P on September 29, 2014, in the Korean Collection for Type Culture, Korea Research Institute of Bioscience and Biotechnology.

The screening method may be performed based on the result of measurement of the amount of lipase produced as a result of measurement of enzymatic activity. However, in order to obtain a transformant capable of producing more effectively transformed lipase, It is preferable to conduct screening using the measurement result and the measurement result of the production amount of lipase together as a measurement standard.

For example, in order to confirm the result of the enzyme activity measurement, a lipase substrate such as triglyceride or pNP-ester is added to a culture solution containing the mutated lipase produced by the transformant, and the ester bond in the substrate is decomposed A method of measuring activity can be used; In order to confirm the result of the production of the mutated lipase, it is possible to use a method of purifying the mutated lipase produced from the transformant and comparing the amount of mutated lipase produced per unit culture liquid.

As another embodiment for accomplishing the above object, the present invention provides a method for producing the mutated lipase of the present invention using the transformant.

Specifically, a method for producing a mutated lipase of the present invention comprises the steps of: (a) culturing the transformant to obtain a culture; And (b) recovering the mutated lipase provided by the present invention from the culture.

As another method, a method for producing the mutated lipase of the present invention comprises the steps of: (a) obtaining a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3; (b) cloning the obtained polynucleotide to obtain an expression vector; (c) introducing the obtained expression vector into a host cell to obtain a transformant; And (d) culturing the transformant, and recovering the mutated lipase comprising the amino acid sequence of SEQ ID NO: 3.

The term "cultivation" of the present invention means a method of growing the microorganism under an appropriately artificially controlled environmental condition. In the present invention, the method for culturing the transformant may be carried out by a method well known in the art. Specifically, the culture can be continuously cultured in a batch process or an injection batch or a repeated fed batch process, as long as it can produce the mutated lipase of the present invention. have.

The medium used for culturing can meet the requirements of a specific strain in an appropriate manner while controlling the temperature, pH and the like under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, and the like. The carbon sources that can be used include glucose and xylose mixed sugar as main carbon sources, and sugar and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, Oils and fats such as oils and the like, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Nitrogen sources that may be used include inorganic sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic substances such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or their decomposition products, defatted soybean cake or decomposition products thereof . These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate and the corresponding sodium-containing salts as a source. Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to these materials, essential growth materials such as amino acids and vitamins can be used.

In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in the culture process in a batch manner, in an oil-feeding manner or in a continuous manner by an appropriate method, but it is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture.

In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. An oxygen or oxygen-containing gas (e.g., air) is injected into the culture to maintain aerobic conditions. The temperature of the culture is usually 27 ° C to 37 ° C, preferably 30 ° C to 35 ° C. The culture is continued until the amount of the produced lipase is maximized. Usually for 10 to 100 hours for this purpose.

In addition, the step of recovering the mutated lipase from the culture can be carried out by a method known in the art. Specifically, the recovering method is not particularly limited as long as the recovered lipase of the present invention can be recovered. Preferably, the recovered lipase is centrifuged, filtered, extracted, sprayed, dried, , Fractional dissolution (for example, ammonium sulfate precipitation), chromatography (for example, ion exchange, affinity, hydrophobicity and size exclusion), and the like.

Preferably, the medium used in the culture of the present invention may be a culture medium containing a carbon source and a nitrogen source necessary for culturing a conventional yeast or a strain, more preferably a non-ionic surfactant, a polysorbate ) (Trade name tween20) or poloxamer (trade name Poloxamer 188), and may be a medium containing a carbon source and a nitrogen source necessary for culturing a conventional yeast or strain.

In addition, the method for producing the mutated lipase provided by the present invention may further comprise the step of purifying the recovered mutated lipase. The purification step may be carried out by any method known in the art such as immunoaffinity chromatography, receptor affinity chromatography, hydrophobic functional chromatography, lectin affinity chromatography, size exclusion chromatography, cation or anion exchange chromatography, high performance liquid chromatography (HPLC) ≪ / RTI > Further, there is a method in which a desired protein is a fusion protein having a specific tag, label, or chelate moiety so that it can be recognized and purified by a specific binding partner or agent. The purified protein may be truncated to the desired protein portion or may remain as such. Cleavage of the fusion protein can result in the desired protein form with additional amino acids in the cleavage process.

In one embodiment of the invention, Candida aentak urticae (Candida The lipase derived from antarctica) strains (Candida antarctica Lipase B, CalB), optimizing the base sequence of the CalB gene so that the CalB gene obtained is efficiently produced in the Pichia pastoris strain, and a polynucleotide encoding the mutated lipase is obtained (FIG. 1) containing the polynucleotide thus prepared was introduced into Pichia pastoris to prepare a transformant. Screening was performed on the transformant thus prepared, and the transformant Transformants with excellent lipase expression efficiency were screened. It was confirmed that the selected transformant can produce CalB having a purity of 90% or more at a yield of 1 g or more per liter of the culture.

In another aspect of the present invention, the present invention provides a method for producing biodiesel using the mutated lipase.

Specifically, the method for producing biodiesel provided by the present invention comprises the steps of (a) culturing a transformant expressing a mutated lipase of the present invention, culturing the transformant, or allowing lipases separated from the culture, Reacting with an alcohol to obtain a reaction product; And (b) recovering the biodiesel from the reactant.

The transformant, culture, or mutated lipase used in the production of the biodiesel can be used in a free form or in an immobilized form. In the case of using the immobilized form, various methods commonly used in the art can be used to immobilize . The immobilizing method is not particularly limited, but a physical method such as an adsorption method and an inclusive method, and a chemical method such as a covalent bonding method and a crosslinking method can be used.

The fat used in the method for producing biodiesel according to the present invention is not particularly limited, but may be natural oil, processed oil, lung oil and the like, and more preferably soy oil, vegetable oil, palm oil, etc. . The alcohol used in the process for producing biodiesel according to the present invention is also not particularly limited, but preferably an alcohol having 2 to 8 carbon atoms can be used, more preferably 2 to 4 carbon atoms Ethanol, methanol, 1-propanol, iso-propanol, 1-butanol, 2-butanol, iso-butanol, ter-butanol and the like can be used most preferably. The alcohol may be supplied using various feeding methods known in the art, and may be supplied in several steps or continuously.

The use of the mutated lipase gene of the present invention can produce a lipase with excellent efficiency from a transformant derived from a Pichia pastoris strain. Therefore, it is widely used in a biological production process of various compounds using lipase including a method of producing biodiesel It can be utilized.

Fig. 1 shows the vector maps of the recombinant expression vectors pPICZaA / CalB-wt and pPICZaA / CalB-OPT for expressing the CalB gene prepared in the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: CalB  Gene Generation

Candida antarctica ), and using the extracted genomic DNA as a template and carrying out PCR using the following primers, a lipase derived from Candida antichatica ( Candida the antarctica Lipase B, CalB) (SEQ ID NO: 1) encoding the CalB gene-wt (SEQ ID NO: 2) was obtained. The PCR conditions were 95 ° C for 5 minutes, 30 cycles (95 ° C for 30 seconds, 56 ° C for 30 seconds and 72 ° C for 1 minute), and 72 ° C for 7 minutes.

Forward primer: 5'-GAATTCAAGCTGCCTTCCGGTTCGGACCCTG-3 '(SEQ ID NO: 5)

(GAATTC: restriction enzyme 1 EcoR I)

Reverse primer: 5'-GCGGCCGCTCAGGGGGTGACGATGCCGGAGCAG-3 '(SEQ ID NO: 6)

(GCGGCCGC: restriction enzyme 2 Not I)

The codon optimization of the CalB-wt gene was performed to efficiently produce lipase from the Pichia pastoris strain so that the CalB-wt gene (SEQ ID NO: 2) obtained above was efficiently produced in the Pichia pastoris strain CalB-OPT gene (SEQ ID NO: 4) was obtained. At this time, the codon optimization was performed using the codon optimization algorithm of the US DNA2.0 (www.dna20.com), and the codon was transformed into the optimal form for expressing the CalB-WT gene in the Pichia pastoris strain. And the CalB-OPT gene was synthesized.

The gene of SEQ ID NO: 4 was expected to be able to express mutated CalB (SEQ ID NO: 3) containing an amino acid sequence different from SEQ ID NO: 1.

Example  2: Production of recombinant expression vector and transformant

The CalB-wt gene and the CalB-OPT gene obtained in Example 1 were treated with restriction enzymes EcoR I / Not I to obtain respective DNA fragments. The obtained DNA fragments were ligated to the polyclonal region of the vector pPICZαA To construct recombinant expression vectors (pPICZaA / CalB-wt and pPICZaA / CalB-OPT) (Fig. 1). At this time, Escherichia coli was used as a host cell for producing a recombinant expression vector.

1 shows a vector map of recombinant expression vectors pPICZaA / CalB-wt and pPICZaA / CalB-OPT for expressing CalB-wt gene and CalB-OPT gene prepared in the present invention.

On the other hand, each of the recombinant expression vectors (pPICZaA / CalB-wt and pPICZaA / CalB-OPT) prepared above was subjected to electroporation transformation using Pichia pastoris pastoris X-33) to prepare respective transformants.

The transformant thus prepared was called " Pichia pastoris KCM-AT ( Pichia pastoris KCM-AT) "and deposited it with the accession number KCTC18326P on September 29, 2014, in the Korean Collection for Type Culture, Korea Research Institute of Bioscience and Biotechnology.

Example  3: Mutated CalB Screening for Optimal Transformants to Produce

Unlike the bacteria in which the expression vector exists as an independent gene and each cell expresses the same introduced gene, even if the same expression vector is introduced into the yeast strain Pichia pastoris, the gene introduced into each transformant May be different. Thus, screening was performed to select transformants that most efficiently produced mutant lipases (CalB-OPT) among the transformants.

Specifically, 0.8% (v / v) v / v was added to BMMY agar medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate, 1.34% YNB, 4x10-5% biotin, 0.5% methanol, 2% agar, ), And then subjected to ultrasonic treatment to prepare an opaque solid medium. The transformant prepared in Example 2 was inoculated into the prepared solid medium and cultured at 30 DEG C for 48 hours. When the inoculated transformant expresses CalB-OPT as it proliferates, the tributyrin contained in the medium is decomposed by the expressed CalB-OPT to form a transparent ring. The amount of the transparent circle As the area or radius increases, 20 strains showing excellent CalB-OPT expression or activity based on the area or radius of the transparent ring were first screened.

Twenty transformants selected through the primary screening were inoculated into 1 ml BMMY liquid medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate, 1.34% YNB, 4x10-5% biotin, 0.5% pH 6.0) and cultured at 30 ° C and 200 rpm in an incubator for 48 hours to obtain a culture. The resulting culture was centrifuged at 4000 rpm for 10 minutes to obtain a culture supernatant. To this supernatant was added pNPB (para-nitrophenyl butyrate) solution (100 μM pNPB in 50 mM tris-HCl, pH 8.0) Lt; / RTI > The pNPB is decomposed by CalB-OPT to produce pNP (para-nitrophenol), which is yellow, as a degradation product. As the expression amount or activity of CalB-OPT increases, the amount of pNP increases. After that, the reaction product was subjected to secondary screening by measuring the absorbance at 405 nm and measuring the highest absorbance of CalB-OPT.

The content of CalB-OPT contained in the cultured product was measured to be 90 (purity), and the purity was 90 % Or more of CalB-OPT was produced at a yield of 1 g or more per liter of the culture.

Microbiology Resource Center KCTC18326P 20140929

<110> KOREA RESEARCH INSTITUTE OF CHEMICAL TECHNOLOGY <120> Process for preparing lipase derived from Candida antarctica          using Pichia pastoris <130> KPA140720-KR <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 317 <212> PRT <213> Artificial Sequence <220> <223> recombinant lypase <400> 1 Leu Pro Ser Gly Ser Asp Pro Ala Phe Ser Gln Pro Lys Ser Val Leu   1 5 10 15 Asp Ala Gly Leu Thr Cys Gln Gly Ala Ser Ser Ser Ser Val Ser Lys              20 25 30 Pro Ile Leu Leu Val Pro Gly Thr Gly Thr Thr Gly Pro Gln Ser Phe          35 40 45 Asp Ser Asn Trp Ile Pro Leu Ser Ala Gln Leu Gly Tyr Thr Pro Cys      50 55 60 Trp Ile Ser Pro Pro Pro Phe Met Leu Asn Asp Thr Gln Val Asn Thr  65 70 75 80 Glu Tyr Met Val Asn Ale Ile Thr Thr Leu Tyr Ala Gly Ser Gly Asn                  85 90 95 Asn Lys Leu Pro Val Leu Thr Trp Ser Gln Gly Gly Leu Val Ala Gln             100 105 110 Trp Gly Leu Thr Phe Phe Pro Ser Ile Arg Ser Lys Val Asp Arg Leu         115 120 125 Met Ala Phe Ala Pro Asp Tyr Lys Gly Thr Val Leu Ala Gly Pro Leu     130 135 140 Asp Ala Leu Ala Val Ser Ala Pro Ser Val Trp Gln Gln Thr Thr Gly 145 150 155 160 Ser Ala Leu Thr Thr Ala Leu Arg Asn Ala Gly Gly Leu Thr Gln Ile                 165 170 175 Val Pro Thr Thr Asn Leu Tyr Ser Ala Thr Asp Glu Ile Val Gln Pro             180 185 190 Gln Val Ser Asn Ser Pro Leu Asp Ser Ser Tyr Leu Phe Asn Gly Lys         195 200 205 Asn Val Gln Ala Gln Ala Val Cys Gly Pro Leu Phe Val Ile Asp His     210 215 220 Ala Gly Ser Leu Thr Ser Gln Phe Ser Tyr Val Val Gly Arg Ser Ala 225 230 235 240 Leu Arg Ser Thr Thr Gly Gln Ala Arg Ser Ala Asp Tyr Gly Ile Thr                 245 250 255 Asp Cys Asn Pro Leu Pro Ala Asn Asp Leu Thr Pro Glu Gln Lys Val             260 265 270 Ala Ala Ala Ala Leu Ala Ala Ala Ala Ile Ala Ala Gly         275 280 285 Pro Lys Gln Asn Cys Glu Pro Asp Leu Met Pro Tyr Ala Arg Pro Phe     290 295 300 Ala Val Gly Lys Arg Thr Cys Ser Gly Ile Val Thr Pro 305 310 315 <210> 2 <211> 954 <212> DNA <213> Artificial Sequence <220> <223> nucleotide <400> 2 ctgccttccg gttcggaccc tgccttttcg cagcccaagt cggtgctcga tgcgggtctg 60 acctgccagg gtgcttcgcc atcctcggtc tccaaaccca tccttctcgt ccccggaacc 120 ggcaccacag gtccacagtc gttcgactcg aactggatcc ccctctctgc gcagctgggt 180 tacacaccct gctggatctc acccccgccg ttcatgctca acgacaccca ggtcaacacg 240 gagtacatgg tcaacgccat caccacgctc tacgctggtt cgggcaacaa caagcttccc 300 gtgctcacct ggtcccaggg tggtctggtt gcacagtggg gtctgacctt cttccccagt 360 atcaggtcca aggtcgatcg acttatggcc tttgcgcccg actacaaggg caccgtcctc 420 gccggccctc tcgatgcact cgcggttagt gcaccctccg tatggcagca aaccaccggt 480 tcggcactca ctaccgcact ccgaaacgca ggtggtctga cccagatcgt gcccaccacc 540 aacctctact cggcgaccga cgagatcgtt cagcctcagg tgtccaactc gccactcgac 600 tcatcctacc tcttcaacgg aaagaacgtc caggcacagg ctgtgtgtgg gccgctgttc 660 gtcatcgacc atgcaggctc gctcacctcg cagttctcct acgtcgtcgg tcgatccgcc 720 ctgcgctcca ccacgggcca ggctcgtagt gcagactatg gcattacgga ctgcaaccct 780 cttcccgcca atgatctgac tcccgagcaa aaggtcgccg cggctgcgct cctggcgccg 840 gcggctgcag ccatcgtggc gggtccaaag cagaactgcg agcccgacct catgccctac 900 gcccgcccct ttgcagtagg caaaaggacc tgctccggca tcgtcacccc ctga 954 <210> 3 <211> 317 <212> PRT <213> Artificial Sequence <220> <223> mutated lypase <400> 3 Leu Pro Ser Gly Ser Asp Pro Ala Phe Ser Gln Pro Lys Ser Val Leu   1 5 10 15 Asp Ala Gly Leu Thr Cys Gln Gly Ala Ser Ser Ser Ser Val Ser Lys              20 25 30 Pro Ile Leu Leu Val Pro Gly Thr Gly Thr Thr Gly Pro Gln Ser Phe          35 40 45 Asp Ser Asn Trp Ile Pro Leu Ser Thr Gln Leu Gly Tyr Thr Pro Cys      50 55 60 Trp Ile Ser Pro Pro Pro Phe Met Leu Asn Asp Thr Gln Val Asn Thr  65 70 75 80 Glu Tyr Met Val Asn Ale Ile Thr Ala Leu Tyr Ala Gly Ser Gly Asn                  85 90 95 Asn Lys Leu Pro Val Leu Thr Trp Ser Gln Gly Gly Leu Val Ala Gln             100 105 110 Trp Gly Leu Thr Phe Phe Pro Ser Ile Arg Ser Lys Val Asp Arg Leu         115 120 125 Met Ala Phe Ala Pro Asp Tyr Lys Gly Thr Val Leu Ala Gly Pro Leu     130 135 140 Asp Ala Leu Ala Val Ser Ala Pro Ser Val Trp Gln Gln Thr Thr Gly 145 150 155 160 Ser Ala Leu Thr Thr Ala Leu Arg Asn Ala Gly Gly Leu Thr Gln Ile                 165 170 175 Val Pro Thr Thr Asn Leu Tyr Ser Ala Thr Asp Glu Ile Val Gln Pro             180 185 190 Gln Val Ser Asn Ser Pro Leu Asp Ser Ser Tyr Leu Phe Asn Gly Lys         195 200 205 Asn Val Gln Ala Gln Ala Val Cys Gly Pro Leu Phe Val Ile Asp His     210 215 220 Ala Gly Ser Leu Thr Ser Gln Phe Ser Tyr Val Val Gly Arg Ser Ala 225 230 235 240 Leu Arg Ser Thr Thr Gly Gln Ala Arg Ser Ala Asp Tyr Gly Ile Thr                 245 250 255 Asp Cys Asn Pro Leu Pro Ala Asn Asp Leu Thr Pro Glu Gln Lys Val             260 265 270 Ala Ala Ala Ala Leu Ala Ala Ala Ala Ile Ala Ala Gly         275 280 285 Pro Lys Gln Asn Cys Glu Pro Asp Leu Met Pro Tyr Ala Arg Pro Phe     290 295 300 Ala Val Gly Lys Arg Thr Cys Ser Gly Ile Val Thr Pro 305 310 315 <210> 4 <211> 954 <212> DNA <213> Artificial Sequence <220> <223> optimized nucleotide <400> 4 ctgccgtcag gttctgaccc ggcctttagc cagccgaagt ctgttctgga tgctggcctg 60 acatgtcagg gtgcaagccc gtcgtccgta agcaaaccaa ttctgcttgt accaggcacg 120 ggcactacgg gcccgcagag ctttgattct aattggattc ccctgtctac ccagcttggg 180 tacacccctt gttggattag cccgcctccc ttcatgctga acgatacaca agtgaatact 240 gagtacatgg tcaacgcaat taccgccctt tatgcgggaa gtggtaacaa taaacttccc 300 gtgctgacat ggagtcaggg gggcctggtg gcacagtggg gattgacgtt tttcccatcg 360 atccgctcga aagttgatcg tctgatggca tttgcgcctg attataaagg cacagtgctc 420 gcggggccat tagatgccct ggctgtgtca gcacctagtg tctggcaaca gacgaccggt 480 tccgcgctga cgaccgccct ccggaacgca ggtggactga cccaaattgt gccgacaacc 540 aacttgtata gcgccaccga cgaaattgtt cagccgcagg tctccaattc gcctctcgat 600 tcaagctatc tgtttaacgg caaaaatgta caggcacagg ctgtttgcgg gccattattc 660 gtcatcgacc atgccggtag cttaacctcc cagttcagtt acgtggttgg tcgctctgcc 720 ctgcgtagta ccacgggcca agcgcgctca gcggactacg gtatcactga ttgcaatccg 780 ttaccggcga atgacctgac tccggaacaa aaggtagcgg ctgcggcttt gttagcgccg 840 gccgctgccg cgattgtggc aggtcctaaa caaaactgtg aaccggatct gatgccctat 900 gcccgtccgt ttgcggtcgg caaacgtact tgctcaggta tcgttacgcc atga 954 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gaattcaagc tgccttccgg ttcggaccct g 31 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gcggccgctc agggggtgac gatgccggag cag 33

Claims (19)

A mutated lipase comprising the amino acid sequence of SEQ ID NO: 3.
The method according to claim 1,
The mutated lipase is called &lt; RTI ID = 0.0 &gt; Candida & antarctica ) strain.
A polynucleotide comprising a base sequence encoding a mutated lipase of claim 1.
The method of claim 3,
Wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4.
An expression vector comprising the polynucleotide of claim 3.
A transformant capable of producing the mutated lipase by introducing the expression vector of claim 5 into a host cell.
The method according to claim 6,
Wherein the transformant is obtained by further performing a step of screening a transformant prepared by introducing an expression vector into the host cell.
8. The method of claim 7,
Wherein the screening is carried out according to a criterion selected from the group consisting of enzyme activity measurement of mutated lipases, production of mutated lipases, and combinations thereof.
9. The method of claim 8,
Wherein the enzymatic activity measurement of the mutated lipase is carried out by measuring an ester-linkage cleavage activity against triglycerides or pNP-esters.
The method according to claim 6,
The host cell may be selected from the group consisting of Pichia pastoris ).
The method according to claim 6,
Wherein said transformant is Pichia pastoris KCM-AT deposited with Accession No. KCTC18326P.
(a) obtaining a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3;
(b) cloning the obtained polynucleotide to obtain an expression vector;
(c) introducing the obtained expression vector into a host cell to obtain a transformant; And
(d) culturing the transformant, and recovering the mutated lipase. 2. The method according to claim 1, wherein the transformant is cultured.
13. The method of claim 12,
Wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4.
13. The method of claim 12,
further comprising the step of purifying the mutated lipase recovered in step (d).
15. The method of claim 14,
The purification can be carried out in a variety of ways, including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic functional chromatography, lectin affinity chromatography, size exclusion chromatography, cation or anion exchange chromatography, high performance liquid chromatography (HPLC) &Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt; combinations thereof.
(a) reacting a mutant lipase of claim 1, a transformant capable of producing the mutated lipase of claim 6, and a culture of said transformant, with a preservative and an alcohol to obtain a reaction product; And
(b) recovering the biodiesel from the reactant.
17. The method of claim 16,
Wherein said mutated lipase, transformant or culture is immobilized.
17. The method of claim 16,
Wherein said oil is a natural oil, a processed oil or a waste oil oil.
17. The method of claim 16,
Wherein the alcohol is an alcohol having from 2 to 8 carbon atoms.

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